We propose to determine whether the structural gene for globin or the neighboring sequences are differently methylated depending whether the gene is expressed or not. For this purpose, DNA will be isolated from the active and inactive gene regions of uninduced and induced Friend cells using DNase II treatment and Mg ions precipitation. Both DNAs will be split with restriction enzymes which are specific for CG regions. The split products will be electrophoresed on agarose gels, transferred to Millipore strips, hybridized to radioactive globin cDNA. Since the cleavage by restriction endonucleases depends on the methylated state of cytosine residues, the autoradiographic profiles should reveal any changes regarding the methylated cytosine residues within or in the vicinity of the globin gene. DNA will be isolated from both the actively transcribed chromatin and inactive chromatin. DNA derived from the actively transcribed chromatin has proven to be a poor methyl acceptor, while DNA isolated from untranscribed chromatin accepts CH3 groups in vitro quite readily. We will attempt to establish that our finding is not an artifact due to the size of DNase II fragments, and propose to confirm these findings by extending them to other types. We find that after DNase II treatment, DNA-methyltransferase activity is almost exclusively associated with the P2, or "inactive", chromatin fraction. Minimal activity is associated with the S2, or "active", chromatin fraction and this activity can be pelleted by high-speed centrifugation. These data indicate that the methyltransferase is bound to the nucleosomes. We will attempt to prove this assumption by showing 1) that DNase I treatment does not liberate any activity, 2) that the activity remains with the nucleosomes after micrococcal nuclease treatment.